Autophagy and Epithelial Cell Fate During Anoikis and 3D Morphogenesis

描述（由申请人提供）：自噬是一种严格调节的溶酶体降解过程，其中细胞在各种形式的胁迫下释放其自身的内容物。在真核细胞中，自噬主要在营养耗尽或应激期间起保护性反应的作用。总的来说，关于这一基本过程如何影响癌症的发生、发展和对治疗的反应，还有很多东西有待了解。在这项提案中，我们将测试自噬通过两种机制影响上皮癌发展的假设-1）增强上皮细胞在ECM破坏诱导的应激和凋亡（失巢凋亡）中存活的能力和2）限制增殖。这一假设是基于使用独特的3D上皮细胞培养系统的初步研究，以检查致癌损伤如何影响腺结构中正常中空腔的形成。在该模型中，管腔形成涉及缺乏与细胞外基质（ECM）直接接触的中央细胞的选择性死亡。最近，我们发现在管腔形成过程中存在高水平的自噬，这表明这一关键过程也有助于上皮细胞的命运。在随后的工作中，我们是第一个建立自噬强烈诱导ECM剥夺（失巢凋亡）。基于这些初步数据，在目标1中，我们将评估自噬是否通过减轻失巢凋亡和3D管腔形成期间ECM脱离的应力来促进细胞存活。此外，我们已经获得了自噬调节分子Beclin/ATG 6（一种已知的肿瘤抑制因子）在3D培养中抑制细胞增殖的初步证据。在目标2中。我们将询问在生长因子撤除和3D形态发生期间是否有其他自噬调节剂（称为ATG）抑制增殖，以便明确地确定自噬可以抑制上皮增殖并鉴定能够抑制肿瘤细胞扩增的新的自噬调节剂。目标3.我们将描述调节自噬如何影响癌基因表达细胞的存活和增殖，以确定如何利用自噬来治疗靶向在已知癌症途径中具有扰动的肿瘤细胞。总的来说，拟议的实验将提供关于自噬在人类癌症发展和进展过程中的确切作用的独特信息。最终，这些研究可能会导致新的治疗策略，通过操纵自噬来防止癌细胞的不受控制的扩张。

DESCRIPTION (provided by applicant): Autophagy is a tightly regulated lysosomal degradation process in which a cell digests its own contents under various forms of duress. In eukaryotic cells, autophagy primarily functions as a protective response during nutrient depletion or stress. Overall, much remains to be learned about how this fundamental process affects cancer initiation, progression and response to therapy. In this proposal, we will test the hypothesis that autophagy influences epithelial cancer development through two mechanisms-1) enhancing the ability of epithelial cells to survive ECM detachment-induced stress and apoptosis (anoikis) and 2) limiting proliferation. This hypothesis is based on initial studies using a unique 3D epithelial culture system to examine how oncogenic insults affect the formation of the normally hollow lumen in glandular structures. In this model, lumen formation involves the selective death of central cells lacking direct contact with extracellular matrix (ECM). Recently, we have discovered high levels of autophagy during lumen formation, indicating that this critical process also contributes to epithelial cell fate. In subsequent work, we are the first to establish that autophagy is strongly induced during ECM deprivation (anoikis). Based on this preliminary data, in Aim 1, we will evaluate whether autophagy promotes cell survival via mitigating the stresses of ECM detachment during anoikis and 3D lumen formation. In addition, we have obtained preliminary evidence that the autophagy regulatory molecule Beclin/ATG6 (a known tumor suppressor) inhibits cell proliferation in 3D culture. In Aim 2. we will interrogate whether other autophagy regulators (called ATGs) inhibit proliferation during growth factor withdrawal and 3D morphogenesis in order to definitively establish that autophagy can restrain epithelial proliferation and to identify new autophagy regulators that are able to inhibit neoplastic cell expansion. In Aim 3. we will delineate how modulating autophagy influences survival and proliferation in oncogene-expressing cells in order to determine how autophagy can be exploited to therapeutically target tumor cells harboring perturbations in known cancer pathways. Overall, the proposed experiments will provide unique information on the precise roles of autophagy during the development and progression of human carcinomas. Ultimately, these studies may lead to new treatment strategies to prevent the uncontrolled expansion of cancer cells via manipulating autophagy.